The adenosine receptor agonist 2-[ p-(2-carboxyethyl)phenylethylamino]-5'- N-ethylcarboxamidoadenosine (CGS 21680) is generally considered to be a selective adenosine A 2A receptor ligand. However, the compound has previously been shown to exhibit binding characteristics that are not compatible with adenosine A 2A receptor binding, at least in brain regions other than the striatum. We have examined binding of [ 3H]CGS 21680 and of antagonist radioligands with high selectivity for adenosine A 1 or A 2A receptors to hippocampus and striatum of mice lacking either adenosine A 1 (A1R (-/-)) or A 2A (A2AR (-/-)) receptors. Both receptor autoradiography and membrane binding techniques were used for this purpose and gave similar results. There were no significant changes in the binding of the A 1 receptor antagonist [ 3H]DPCPX in mice lacking A 2A receptors, or in the binding of the A 2A receptor antagonists [ 3H]SCH 58261 and [ 3H]ZM 241385 in mice lacking A 1 receptors. Furthermore, [ 3H]CGS 21680 binding in striatum was abolished in the A2AR (-/-), and essentially unaffected in striatum from mice lacking A 1 receptors. In hippocampus, however, binding of [ 3H]CGS 21680 remained in the A2AR (-/-), whereas binding was virtually abolished in the A1R (-/-). There were no adaptive alterations in A 2A receptor expression in this region in A1R (-/-) mice. Thus, most of the [ 3H]CGS 21680 binding in hippocampus is dependent on the presence of adenosine A 1 receptors, but not on A 2A receptors, indicating a novel binding site or novel binding mode.